General Discussion and Future Perspectives 8 191 What have we learned? This thesis started by presenting an analogy between the studying (quantification) of bees and EVs to infer environmental or cellular status. In both cases, specific tools are needed to fulfil this purpose. For example, bee-keepers may count the number of inter-frames covered with adult bees from above the hive body to estimate the adult worker population size of a given colony 41. In this thesis we developed and validated a standardized assay for the direct detection of single EVs in complex biofluids using IFCM. Although many techniques for EV characterization and quantification have been (and continue to be) developed, the method as presented in this thesis represents an important step towards the standardized reporting of analytical parameters such as size and fluorescence for single EVs. The ability of our protocol to detect even low abundant subpopulations of EVs without performing EV isolation demonstrates its potential to become a key tool in the EV-field, and we expect that EV IFCM may one day be used in the diagnosis, prognosis and monitoring of health and disease. FUTURE DIRECTIONS Is the technology ready for the clinic? Single EV FC is considered the holy grail in EV characterization as it has the potential to detect, size, and phenotype thousands to millions of single EVs per minute 14. The work described in this thesis has identified IFCM as a suitable platform for single EV detection. We believe that IFCM could serve as a diagnostic tool in the clinic, especially given its ability to detect low abundant EV subpopulations as demonstrated in this thesis. However, there are some areas in which the technique can be further improved. In this thesis, we calibrated the light scattering intensities as detected by our IFCM to reflect particle sizes (through Mie theory). However, the obtained degree of correlation between predicted and measured scatter intensities for polystyrene (PS) beads (R2 = 0.91) was not on the level of conventional, state-of-the art FCMs (R2 ≥ 0.99). This implies an approximate 8% error when estimating the size of EVs, which, in turn, impairs reproducibility. Thus, the calibration of light scattering signals into particle size should be further optimized for IFCM. Ongoing efforts are currently examining whether light scattering signals generated by a 405-nm laser may lead to improved scatter-to-size resolutions, using PS beads to calibrate light scatter signals and hollow organosilica beads (HOBs - with refractive resembling that of EVs 42) to validate these calibrations 14.
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